Defects in many genes with roles in DNA break repair are associated with a striking predisposition to cancer development. One of the most extreme cancer risks is associated with Bloom syndrome (BS) - a chromosome breakage disorder caused by mutations in the RecQ-like DNA helicase BLM. RecQ-like helicases and their role in regulating recombinational DNA repair are conserved from bacteria to humans. Besides BS, defects in RecQ-related genes cause Werner syndrome and Rothmund-Thompson syndrome, which are characterized by accelerated aging and/or increased cancer risk. In addition to BS-associated mutations, 93 missense mutations in the human BLM gene have been reported, but it is unknown which, if any, affect BLM function. It has also been suggested that single nucleotide polymorphisms (SNPs) in introns of BLM that have been associated with higher cancer risk may be linked to coding SNPs in exons of BLM. Using a yeast Sgs1-BLM chimera, we have identified coding SNPs that impair BLM function. They include hypomorphic mutations that define a new class of BLM alleles, not associated with BS, that may increase genome instability, cancer risk and other BS-associated symptoms. One objective of this proposal therefore is to determine the effect of coding SNPs throughout the BLM gene on chromosome stability, DNA break repair and the DNA-damage response, and identify their biochemical defects. In contrast to the helicase core, the >600-residue long N- terminal tails of BLM and the related yeast helicase Sgs1 are disordered and not conserved at the sequence level. They have therefore been refractory to conventional approaches to elucidate their function. It is our hypothesis that the function of the long tails of Sgs1 and BLM arises from structural elements, embedded in disorder, that serve as molecular recognition elements for binding proteins. To test this hypothesis we have designed an approach that combines computational prediction of disorder and interactivity, structure analysis by nuclear magnetic resonance (NMR) spectroscopy, and proline mutagenesis to identify these structural elements and elucidate their importance for BLM and Sgs1 function. Specifically we will (1) use a population- based mutational approach to identify and characterize novel functional motifs in BLM; the ability of BLM variants to rescue high sister-chromatid exchange, double-strand-break-repair defects and hypersensitivity to DNA-damaging agents will be assessed; (2) identify biochemical defects of functionally impaired BLM variants by assessing ATPase, DNA binding, annealing and unwinding activities, and (3) determine disorder-function relationships in the N-terminal tails of Sgs1 and BLM using a combination of (a) NMR to identify regions that are dynamically constrained and may adopt interaction-prone a-helices, (b) proline mutagenesis to disrupt the structural motifs, and (c) functional analysis of novel separation-of-function alleles of SGS1 and BLM in vivo. New insights into function and connectivity of BLM and Sgs1 will elucidate the mechanisms of hyper- recombination and chromosome instability in Bloom syndrome and, generally, in human cancers.